Measuring transport dynamics in temporally and spatially heterogeneous nano- and micro-scale systems (e.g., cargo vesicles in vivo) requires high-resolution and high-fidelity concurrent tracking of individual objects over large fields of view. Laser scanning confocal or multiphoton fluorescence microscopies are powerful approaches for such imaging and tracking. Yet, suitable systems, particularly those with spinning discs or resonant galvanometers, are often expensive, specialized, and closed “black boxes” that hinder operator innovation. These systems must, for example, have rapid scanning rates to avoid the spatiotemporal artifacts in the measured sample dynamics that arise from the laser scanning spatial response function.
Consider a raster laser scanning multiphoton fluorescence microscopy imaging process with a raster scanner (e.g. resonant or non-resonant galvanometer) and non-descanned detection: sweeping an excitation spot or pattern over a sample area in the conjugate plane of an array detector produces an image. The exposure time of that detector must be synchronized to the period of the raster scanner drive waveform. In particular, the exposure time must be synchronized to the period of the drive waveform for the slow axis. This introduces an asymmetry, as the fast axis is continuously sampled, while the slow axis is discretely sampled. As a result, raster scanning utilizes only a portion of the possible scanning power spectral bandwidth, limiting (non-resonant) full-field imaging rates to a few frames per second. Furthermore, no information is available about an area of a sample while the excitation beam is elsewhere. For processes with timescales comparable to or faster than the full frame rate of the raster scanner, especially those with wide fields of view, temporal and spatial aliasing artifacts result, due to the inability of the system to sufficiently resolve events over the entire sample.
To improve scanning efficiency, some have proposed multifocal multiplexing using two-dimensional patterns of excitation spots dithered within the periodic bounds of the array. Initial implementations of multiphoton multifocal microscopy (MMM), for example, utilized microlenses, cascaded beamsplitter arrays, and low-spot-density diffractive optical elements (DOE) to generate excitation patterns. DOE beamsplitters offer compactness, stability, efficiency, and uniformity. DOE throughput (˜75%) is similar to microlens arrays without pinholes and allows producing a large number of beams (˜100) with uniform intensity (<5% peak-to-peak variation). However, previous designs relied on conventional raster scanning and lead to oversampling at the edges of each unit cell due to the finite mechanical response of the scanner. Hence, the resulting images exhibited an undesirable, gridwork appearance. It is, therefore, desirable to maintain flexibility for the utilization of random access scanning modes, such as have been recently employed for measurement of intracellular transport, but in a system capable of accurate imaging over an entire sample region and without the bandwidth limitations of conventional raster scanners.